Cholesterol is an abundant substance in animals because it has an essential function in the membranes of animal cells. Cholesterol is almost completely insoluble in aqueous solution. Within the body other than in membranes, therefore, cholesterol must be carefully segregated from exposure to aqueous environments. Therefore, in multicellular organisms, cholesterol is transported about the body packaged within the hydrophobic cores of plasma lipoproteins. The various plasma lipoproteins in humans have been classified into four major classes, principally by buoyant density. The four major classes are very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) low density lipoprotein (LDL), and high density lipoprotein (HDL). A fifth class of lipoprotein is chylomicrons, which occur only after feeding. The most abundant cholesterol-carrying lipoproteins in human plasma are of the LDL class, and high levels of LDL circulating in blood plasma are associated with arteriosclerosis and coronary dysfunction. Hence, the mechanisms which purge LDL from the blood stream have been subject to significant study.
In higher animals, LDL is scavenged from the blood stream by receptors located on the cell membranes of animal cells. The receptors are known as the LDL receptor. The structure of the LDL receptor has been generally described, and the gene coding for the production of the receptor has been isolated and sequenced. Descriptions of the LDL receptor, its structure, and its general functionality in interaction with other cellular processes can be found in Schneider, Bio. Et. Biophys. Acta., 988, pages 307-317 (1989) and Hobbs, et al., Annu. Rev. Genet., 24, pages 133-170 (1990). The LDL receptor protein is a cell surface glycoprotein that regulates plasma cholesterol by mediating endocytosis of lipoproteins. The human LDL receptor is a protein of 860 amino acids encoded by a gene which actuates the transcription of an mRNA of 5.3 kb in length. The mRNA of the human LDL receptor gene includes a long 3' untranslated region, as well as a signal peptide which actuates transport of the protein to the plasma membrane, and which is cleaved from the mature form of the protein.
The LDL receptor protein has been characterized as having generally five different domains or regions. One region, referred to as the ligand binding domain, is generally associated with the affinity of the receptor for the two known proteins which are bound by the receptor, ApoB-100, the 550 kd glycoprotein that is the predominant protein of LDL, and ApoE, a 34 kd protein found in multiple copies in IDL and a subclass of HDL. Other domains in the mature LDL receptor protein include an EGF (epidermal growth factor) precursor homology region which, while not specifically necessary for ligand affinity, appears to play a role in the presentation of the LDL receptor to lipoproteins external to the cell. The third domain of the LDL receptor is a region containing clustered O-linked sugar chains. A membrane spanning region of 22 amino acids, of high hydrophobicity, is responsible for the membrane bound nature of the protein. At the carboxyl end of the protein is a domain of 50 amino acids, which extends into the cytoplasm.
While the human LDL receptor gene and the human LDL receptor protein have been studied extensively, this study has been a difficult and, at times, an arduous process. One of the reasons for the difficulty of this study is the inability of researchers to isolate and utilize large quantities of the LDL receptor protein itself. While the protein is produced in significant quantities in animal cells, it is membrane bound and presumably insoluble in aqueous solution. Hence, it is quite difficult to isolate the receptor protein from mammalian tissues in any significant quantity. In addition, since the affinity for membranes is quite strong, it is difficult to express the human LDL receptor protein in heterologous hosts and isolate pure quantities of protein, due to the strong tendency of the protein to associate with membranes in the expression system. In addition, the protein has a complex three dimensional structure, particularly in the ligand binding domain, which is cysteine-rich and includes multiple disulfide bonds. Since the three dimensional structure of the protein is essential for the affinity of the receptor to LDL, production of the protein in hosts which do not properly process proteins subsequent to translation to result in a correct three dimensional structure would not give rise to functional receptor protein.